The present disclosure relates, in various exemplary embodiments, generally to compositions and methods for enabling adhesion, proliferation and self-renewal maintenance of pluripotent, or undifferentiated, stem cells in vitro. These stem cells include embryonic stem cells, such as murine or human, induced pluripotent stem cells, and bone marrow stem cells.
A stem cell is an undifferentiated cell from which specialized cells are subsequently derived. Embryonic stem cells and induced pluripotent stem reprogrammed from differentiated cells possess extensive self-renewal capacity and pluripotency with the potential to differentiate into cells of all three germ layers. They are useful for therapeutic purposes and may provide unlimited sources of cells for tissue replacement therapies, drug screening, functional genomics and proteomics. (Skottman, H., Dilber, M. S., and Hovatta, O. (2006); The derivation of clinical-grade human embryonic stem cell lines; FEBS Lett 580, 2875-2878).
Murine pluripotent embryonic cells can be maintained in a pluripotent state in in vitro cell culture conditions in the presence of Leukemia Inhibitory Factor (LIF). (Williams R L, Hilton D J, Pease S, Willson T A, Stewart C L, Gearing D P, Wagner E F, Metcalf D, Nicola N A, Gough N M (1988); Myeloid leukemia inhibitory factor maintains the developmental potential of embryonic stem cells, Nature 1988 Dec. 15; 336(6200):684-7).
Additionally, murine pluripotent embryonic cells can be maintained in a pluripotent state in vitro when cultured on mouse embryonic fibroblasts as feeder cells. Human embryonic cells also require feeder cells for maintenance in a pluripotent state in vitro or differentiation inhibitors like Noggin and/or high doses of basic fibroblast growth factor (FGF) when cultured on Matrigel™ (see for review: Skottman, H., Dilber, M. S., and Hovatta, O. (2006); The derivation of clinical-grade human embryonic stem cell lines; FEBS Lett 580, 2875-2878). However, the use of feeder cells has a number of drawbacks. For example, feeder cells can contain pathogens, such as viruses that can infect the stem cells (Hovatta, O., and Skottman, H. (2005); Feeder-free derivation of human embryonic stem-cell lines; Lancet 365, 1601-1603; Skottman, H., Dilber, M. S., and Hovatta, O. (2006); The derivation of clinical-grade human embryonic stem cell lines; FEBS Lett 580, 2875-2878).
Feeder-free systems that support human embryonic stem cell self-renewal require either i) Matrigel™ (Richards, M., Fong, C. Y., Chan, W. K., Wong, P. C., and Bongso, A. (2002); Human feeders support prolonged undifferentiated growth of human inner cell masses and embryonic stem cells; Nat Biotechnol 20, 933-936); (Xu, C., Inokuma, M. S., Denham, J., Golds, K., Kundu, P., Gold, J. D., and Carpenter, M. K. (2001); Feeder-free growth of undifferentiated human embryonic stem cells; Nat Biotechnol 19, 971-974); (Xu, R. H., Peck, R. M., Li, D. S., Feng, X., Ludwig, T., and Thomson, J. A. (2005); Basic FGF and suppression of BMP signaling sustain undifferentiated proliferation of human embryonic stem cells; Nat Methods 2, 185-190); or, ii) mouse feeders-derived extracellular matrix (Klimanskaya, I., Chung, Y., Meisner, L., Johnson, J., West, M. D., and Lanza, R. (2005); Human embryonic stem cells derived without feeder cells; Lancet 365, 1636-1641) as adhesive substrata. However, these coatings are of xenogenic origin and therefore cannot be used in clinics according to FDA requirements (Hovatta, O., and Skottman, H. (2005); Feeder-free derivation of human embryonic stem-cell lines; Lancet 365, 1601-1603). These coatings also fail to fulfill criteria of defined system and non-immunogenicity, importance of which is discussed in (Hovatta, O., and Skottman, H. (2005); Feeder-free derivation of human embryonic stem-cell lines; Lancet 365, 1601-1603; Skottman, H., Dilber, M. S., and Hovatta, O. (2006); The derivation of clinical-grade human embryonic stem cell lines; FEBS Lett 580, 2875-2878).
During mammalian embryonic development, a fertilized oocyte first divides into two cells, followed by another cell duplication to generate a four-cell embryo. At the four-cell stage, the embryonic cells are bound together with the help of cell membrane proteins and also the molecules of a new connective tissue (extracellular matrix). The first extracellular matrix molecules to appear are basement membrane proteins, such as laminin and proteoglycan (Cooper, A. R., and MacQueen, H. A. (1983); Subunits of laminin are differentially synthesized in mouse eggs and early embryos; Dev Biol 96, 467-471) (Dziadek, M., and Timpl, R. (1985); Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells; Dev Biol 111, 372-382). Subsequently, the embryonic cells start to differentiate into the three germ cell layers; ectoderm, endoderm and mesoderm, with initiation of morphogenesis. The extracellular matrix molecules, such as laminins are responsible for interactions with cell surface receptors, thus regulating cell behavior such as adhesion, proliferation, migration and differentiation (Colognato, H., and Yurchenco, P. D. (2000); Form and function: the laminin family of heterotrimers; Dev Dyn 218, 213-234), while other extracellular matrix components such as collagens of types I, II, III or IV primarily serve a mechanical supportive function (Aumailley, M., and Gayraud, B. (1998); Structure and biological activity of the extracellular matrix; J Mol Med 76, 253-265).
Extracellular matrix derived from murine fibroblasts, in combination with soluble differentiation inhibitors may be an adequate replacement for feeder cells (Klimanskaya, I., Chung, Y., Meisner, L., Johnson, J., West, M. D., and Lanza, R. (2005); Human embryonic stem cells derived without feeder cells; Lancet 365, 1636-1641), which demonstrates the critical role of extracellular matrix molecules. Laminins are large trimeric extracellular matrix proteins that are composed of alpha, beta, and gamma chains. There exist five different alpha chains, three beta chains and three gamma chains that in mouse and human tissues have been found in at least fifteen different combinations (Colognato, H., and Yurchenco, P. D. (2000); Form and function: the laminin family of heterotrimers; Dev Dyn 218, 213-234); (Aumailley, M., Bruckner-Tuderman, L., Carter, W. G., Deutzmann, R., Edgar, D., Ekblom, P., Engel, J., Engvall, E., Hohenester, E., Jones, J. C., et al. (2005); A simplified laminin nomenclature; Matrix Biol 24, 326-332). These hetertrimeric molecules are termed laminin-1 to laminin-15 based on their historical discovery, but an alternative nomenclature describes the isoforms based on their chain composition, e.g. laminin-111 (laminin-1) that contains alpha-1, beta-1 and gamma-1 chains (laminin nomenclature: (Aumailley, M., Bruckner-Tuderman, L., Carter, W. G., Deutzmann, R., Edgar, D., Ekblom, P., Engel, J., Engvall, E., Hohenester, E., Jones, J. C., et al. (2005); A simplified laminin nomenclature; Matrix Biol 24, 326-332)).
The different isoforms are developmentally regulated and have tissue specific locations and functions. LN-111 (previously named laminin-1 or laminin) is present in the early embryo and later in certain epithelial cells and murine EHS sarcoma, but otherwise it is a rare isoform in vivo. LN-511 (LN-10) is the most common form found in basement membranes of the early embryo and most adult tissues. Importantly, it is found in the extracellular matrix between cells of the inner cell mass of blastocysts. Laminins are cell type-specific mediators regulating cell adhesion, proliferation, migration, and resistance to apoptosis. Mutations in most laminin chains result in severe pathologies and mortality.
Most laminin isoforms, except for laminin-111, are difficult to extract and purify in native forms from tissues due to extensive crosslinking with other laminins or other macromolecules. Only recently human/mouse hybrid LN-111 (LN-1), and human LN-211 (LN-2), LN-332 (LN-5) specific for epithelial basement membranes, LN-411 (LN-8) common in vascular basement membranes and the ubiquitous LN-511 (LN-10) have been successfully produced as recombinant proteins. It is also possible to isolate some laminin isoforms like LN-332 (LN-5) from cultured cells, but only in low quantities.
There is a need to develop defined feeder cell free in vitro culture systems for establishment and maintenance of undifferentiated mammalian embryonic stem cells, induced pluripotent stem cells, as well as bone marrow stem cells. A major problem of embryonic stem cell cultures is the lack of appropriate surface coatings, particularly regarding human embryonic stem cells. Although defined xeno-free embryonic stem cell culture media have successfully been developed for human embryonic stem cells, researchers are still looking for defined xeno-free non-immunogenic culture plate coatings that do not induce cellular differentiation. Adhesive surface coatings are usually based on various combinations of extracellular matrix proteins, such as laminin-111, collagen IV, gelatin, fibronectin, or Matrigel™, most of which are undefined or not human. For instance, successfully used coatings, such as Matrigel™ prepared from the mouse EHS sarcoma, or extracellular matrix derived from mouse embryonic fibroblasts, are of undefined non-reproducible composition and derived from animal sources. Therefore, they are not applicable for clinical purposes.
Laminins that are central components of basement membranes (BM) are the first extracellular matrix molecules to contact cells in the early embryo. Expression of laminin chains has been shown to occur already at the 2-4 cell stage in mouse embryos. One of the laminins, laminin-511, contacts the inner cell mass of blastocysts which is the natural origin of embryonic stem cells. Laminins have been shown to influence cellular differentiation and migration, in addition to promoting adhesion and proliferation, while some other extracellular matrix molecules like collagens primarily provide adhesion and structural support functions. Thus laminins may be useful for culturing embryonic stem cells in vitro, as they are a natural part of the niche for their origin in vivo.
Notwithstanding the above, there continues to be a need for providing compositions and methods for culturing and growing embryonic stem cells. In this regard, providing compositions and methods for enabling the proliferation and survival of pluripotent stem cells in vitro without use of differentiation inhibitory agents such as LIF or feeder cells would be advantageous.